PROJECT 4 FSH-Patient Derived IPS Cells to Study the Developmental Regulation of DUX4 Transcription The D4Z4 repeat array with multiple copies of the intronless DUX4 gene likely developed as a consequence of retrotransposition of an ancestral gene expressed in ES cells. Our recent publication and preliminary data shows that transcripts from D4Z4 are readily detectable in ES cells making them an important cell type for the study of DUX4 transcriptional regulation. Our preliminary data are consistent with the idea that DUX4 is expressed constitutively in ES cells and its regulation is largely achieved by transcriptional silencing as cells differentiate into other tissue types. Our hypothesis is that FSHD is caused by sequence variations in D4Z4 that disrupt this silencing process with unique toxicity to muscle cells. Our long term goal is to define the normal and pathogenic transcription patterns during development, and identify sequences responsible for the altered pattern of gene expression in FSHD.
In Aim 1 we describe experiments to develop and characterize IPS cells from controls and FSHD-affected patients, and determine if epigenetic markers of D4Z4 regulation seen in ES cells are recapitulated in IPS cells.
In Aim 2, experiments are described to identify transcriptional enhancer and silencing regions that are presumably the targets of developmental regulation of the D4Z4 locus, and therefore will be important targets for therapeutic intervention. We will determine how transcriptional regulation is altered when these areas are removed or mutated using human and mouse developmental models.
In Aim 3 we will modify cells from FSHD-affected patients to produce isogenic clones with homozygous D4Z4 genotypes and quantify transcription differences that result as a consequence of these modifications. In so doing we will produce myoblasts from FSHD patients that contain only non-pathogenic an-ays and with advances in IPS cell development, could be used for autologous transplantation.

Public Health Relevance

The significance of these studies is that an understanding of normal and pathogenic developmental patterns of DUX4 transcriptional regulation will provide a model for disease pathophysiology and help identify therapeutic targets for treatment.